Both monoacetylhydrazine and diacetylhydrazine are metabolites of isoniazid in man and rat. Monoacetylhydrazine has previously been shown to be a potent hepatotoxin in rats, and, based on several studies, oxidation of this compound by microsomal P-450 enzymes, appears to cause the serious hepatitis observed in some patients receiving isoniazid therapy. However, diacetylhydrazine, which is formed by acetylation of monoacetylhydrazine, is found in human and rat urine samples in greater amounts. Therefore, in experiments using both monoacetyl-hydrazine and diacetylhydrazine, we tested for hepatotoxicity and covalent binding of these metabolites in rats. As determined previously, monoacetylhydrazine causes liver necrosis in phenobarbital pretreated rats and covalently binds to hepatic tissue both in vivo and in vitro. In contrast, diacetylhydrazine did not produce liver lesions and did not significantly bind to hepatic tissue in vivo or in vitro. Potential chronic hepatotoxicity in rats of both monoacetylhydrazine and diacetylhydrazine is now being studied.